ElectroLigase®

ElectroLigase® combines T4 DNA Ligase and an optimized, ready-to-use 2X reaction buffer containing a proprietary ligation enhancer and no PEG. This combination is specifically formulated to promote robust ligation of all types of DNA ends (e.g., blunt, sticky, TA). It is directly compatible with electrocompetent cells used for transformation by electroporation, without desalting or purification.

Traditional Cloning Workflow

Overview of PCR Cloning

Ordering Information

ElectroLigase™ combines T4 DNA ligase and an optimized, ready-to-use 2X reaction buffer containing a proprietary ligation enhancer and no PEG. This combination is specifically formulated to promote robust ligation of all types of DNA ends (blunt, sticky, TA). It is directly compatible, without desalting or purification, with electrocompetent cells used for transformation by electroporation. No thawing of the buffer is required as it maintains a liquid state during storage at -20°C*, thereby simplifying reaction set-up. By providing an optimized ratio of enzyme and buffer components, users are able to rapidly ligate all types of DNA ends applying a short incubation time at room temperature. Ligations for subcloning can be carried out in small volumes with low concentrations, allowing users to conserve precious DNA samples. These reactions can be pipetted directly, without purification or dilution, to transform many strains of electrocompetent E. coli**.

* Freezers vary in their actual internal temperatures. Our testing demonstrates that the enzyme and buffer remain liquid at -20°C.

**ElectroLigase is also compatible with chemically competent strains of E. coli. Performance is generally around 50% efficiency, when compared to the Blunt/TA Ligase Master Mix (NEB #M0367 ).

Ligation reactions containing equal amounts (20 ng vector
and 3-fold molar excess of insert) of blunt- (A), T/A (B), or sticky-end (C)
vector/insert pairs were set up using ElectroLigase and incubated for the times
shown. After heat inactivation, 2 μl of each reaction were withdrawn and
directly used to transform NEB 10-beta Electrocompetent E. coli (NEB #C3020). 50 μl aliquots of the outgrowth
(diluted, in some cases) were plated onto selective plates and incubated
overnight at 37°C. Colonies were counted, adjusted for plating dilution, and
graphed.

Storage Temperature

Heat Inactivation

Companion Products

Cells: Competent cells can vary by several logs in their competence. Perceived ligation efficiency directly correlates with the competence of the cells used for transformation. Always transform uncut vector as a control for comparison purposes.

DNA: Purified DNA for ligations can be dissolved in dH2O (Milli-Q® water or equivalent is preferable); TE or other dilute buffers also work well. For optimum ligation, the amount of vector DNA should be 20–100 ng and the insert should be added at a 3-fold molar excess. For ligation volumes greater than 11 μl, increase the volume of ElectroLigase Reaction Buffer accordingly. Insert:vector ratios between 2 and 6 are optimal for single insertions. Ratios below 2:1 result in lower ligation efficiency. Ratios above 6:1 promote multiple inserts. If you are unsure of your DNA concentrations, perform multiple ligations with varying ratios.

Time and Temperature: Most ligations performed using ElectroLigase reach an end point at 60 minutes or less when performed between 4–37°C. Incubation beyond this time provides no additional benefit. Our recommendation for a 25°C (room temperature) incubation was chosen after evaluation of performance at 4°C, 16°C, 25°C, and 37°C. Most conditions reached at least 50% performance within 30 minutes.

Biology: Some DNA sequences are not easy to clone. Sequences that form structures, including inverted and tandem repeats, are selected against by E. coli. Some recombinant proteins are not well tolerated by E. coli and can result in poor transformation or small colonies

The Product Summary Sheet, or Data Card, includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name the majority of these document files: [Catalog Number]Datasheet-Lot[Lot Number]. For those product lots not listed below, please contact NEB at info@neb.com or fill out the Technical Support Form for appropriate document.)

Quality Control Assays

Quality Control tests are performed on each new lot of NEB product to meet the specifications designated for it. Specifications and individual lot data from the tests that are performed for this particular product can be found and downloaded on the Product Specification Sheet, Certificate of Analysis, data card or product manual. Further information regarding NEB product quality can be found here.

This product is covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB).

While NEB develops and validates its products for various applications, the use of this product may require the buyer to obtain additional third party intellectual property rights for certain applications.

For more information about commercial rights, please contact NEB's Global Business Development team at gbd@neb.com.

This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.

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